(Zygoptera: Calopterygidae)

Transcription

1 Odonatologica 36(4): A revised molecular phylogeny of the Calopteryginae (Zygoptera: Calopterygidae) H.J. Dumont,A. Vierstraete and J.R. Vanfleteren Department of Biology, Ghent University, Ledeganckstraat 35, B-9000 Gent, Belgium Henri.Dumont@UGent.be Received June 16, 2007 / Reviewed and AcceptedAugust 2, 2007 An updated version of an ITS-based phylogeny of the Calopteryginae, using sequences of 31 ingrouptaxa, is given. The subfamily consists of 3main clades, each with 2 subclades. Only clade I {Calopteryx s. is s.) not exclusively Asian but extends to Europe and North America. Inthe East-Asian clade 2, the genus Matrona is found to be descended from an Atrocalopteryx-like ancestor. Several so-called South-East Asian Calopteryx probably either belong to Atrocalopteryx or to as yet unnamed genera near Atrocalopteryx. Archineura consists of 2 spp., limited to China and Indo-China, and is rather basal to clade 3, The subclade Neurobasis-Matronoides is worthy of further analysis. INTRODUCTION The dragonfly cohort Caloptera, composed of seven families, has recently been subjected to a phylogenetic analysis using the nuclear ribosomal genes 18S, 5.8S, and the internal transcribed spacers ITS1 and IRS2 (DUMONT et al., 2005). From this analysis, it appeared that the 5.8 and 18S genes in this group evolve at such a slow rate that almost all phylogenetically resolving power lies with the ITSses. The Calopterygidae and the Haeterinidae constitute the two most speciose families of the cohort, and within the Calopterygidae, the largest subunit is the Calopteryginae, with currently about nine genera recognized. In this subfamily, DUMONT et al. (2005) created a new genus, Atrocalopteryx, based on the position of a single species examined, A. atrata, distributed from the coastal fringeof Eastern Siberiato north-easternchinaand Korea. However, this does not mean that the genus is monotypic: several species orginally described

2 366 H.J. Dumont, A. Vierstraete & J.R. Vanfleteren in Calopteryx and occurring further south in China and Vietnam-Laos, are suspected to belong here, but none was so far available for molecular analysis. A Chinese endemic, Archineura incarnata, was found to cluster with Matrona and Atrocalopteryx, a surprising result, since by habitus and morphology, this taxon looked closely related to yet another calopterygine, Leucopteryx haeterinoides, from Laos and Vietnam. The quotes suggest the uncertainty that surrounded (and still surrounds) the generic name Leucopteryx, since classical morphology led to the expectation that haeterinoides was a true Archineura. Shortly after the publication of DUMONT et al. (2005), the suspicion about the placement of Archineura incarnata was confirmed, when it was found that this taxon had been identifiedand sequenced using a femalethat in fact belonged to Table I Alphabeticallist of taxa and specimens examined Geographical origin Collector Archineura hetaerinoides helaerinoides Laos M. Hamalainen Archineura incarnata incarnala Nankunshan, Guangdong,China Hoping Boping Han Atrocalopteryx atrata Dokigawe River, Japan K. Inoue Atrocalopteryx atrocyana atrocyam Nanling, Guangdong, China K.D.P. Wilson Atrocalopteryx irocalopleryx atrocyana Tian Men Gao, Guangdong, China H.J. Dumont Calopteryx Calopteryx" coomani Vietnam M. Hamalainen Calopteryx aequabilis Wisconsin, USA S.W. Dunkle Calopteryx amata New Brunswick, Canada S.W, S.W. Dunkle Calopteryx cornelia Yuragawe River, Japan K. Inoue Calopteryx exul Ifrane, Atlas, Marocco H.J. Dumont Calopteryx haemorrhoidalis Ifrane, Atlas, Morocco H.J, H.J. Dumont Calopteryx maculata Oklahoma, USA S.W. Dunkle Calopteryx virgo virgo Laon, France H.J. Dumont Calopteryx xanthostoma Echo modesta Argens, Chateauvert, France Kanchanaburi,Thailand M. Papazian M. Hamalainen Matrona Malrona basilaris Omei Shan, Sichuan, China Su Rong Matrona basilaris Nankunshan, S. Guangdong, China H.J. Dumont Matrona basilaris Tian Men Gao, C. Guangdong,China H.J. Dumont Matrona basilaris "basilaris" Hainan Island, China (2005) K.D.P. Wilson H.J. Dumont Matrona basilaris "basilaris" Hainan Island, China (2006) Matrona cyanoptera Wash, Taipei,Taiwan W.C. Yeh Matrona nigripecta Chiang Mai, Thailand M. Hamalainen Matronoides cyaneipennis Mt. Kinabalu, Borneo M, M. Hamalainen Mnais andersoni Chiang Mai, Thailand M. Hamalainen Mnais mneme mneme Hainan, China K.D.P. Wilson Neurobasis Neurohasis chinensis Kanchanaburi,Thailand M. Hamalainen Neurobasis chinensis Asan lake, N, N. India H.J. Dumont Hainan Island, China K.D.P. Wilson Neurobasis Neurohasis chinensis Psolodesmus dorothea South Taiwan W.C, W.C. Yeh W.C. Yeh Psolodesmus mandarinus North Taiwan K, Watanabe Psolodesmus mandarinus kuroiwae Mt. Omoto, Ishikagi, Japan

3 The Revised molecular phylogeny of Calopteryginae 367 Matronaor Atrocalopteryx. Thus, new material was collected from Guangdong, South China, to rectify this error. In the same general area (South China) and in Vietnam, more fresh specimens belonging to Matrona suspected to belong to Atrocalopteryx were collected as well, inorder to substantiate better these subdivisions of the calopterygine clade. Here, we sequence and analyse the internal transcribed spacers and intervening 5.8 S rdna of these additional (seven in all) insects, with an aim at obtaining an improved phylogeny of the subfamily. Beside the Archineura, three were suspected to belong to Atrocalopteryx (two to the taxon atrocyana, and one to coomani), and three to Matrona. They were inserted in a tree of Calopteryginae composed of 31 ingroup taxa, plus three outgroup taxa of the zygopteran families Chlorocyphidae, Diphlebiidae, and Megapodagrionidae. The origin and collectors of the specimens analysed is given in Table I. MATERIAL AND METHODS DNA EXTRACTION, PCR AMPLIFICATION AND SEQUENCING - The origins of the samples used in this study are listed in Table I. All were males that had been fixed and preserved in 70-80% ethanol immeditalyupon collection ion the field. In the laboratory, muscular tissue was isolated from the synthorax and genomic DNA was extracted, using the protocol of the Puregene DNA isolation kit type D-5000A (Centra Systems Inc., BlOzym, Netherlands),or using a modified CTAB protocol (KOCHER et at, 1989), Briefly, muscle tissue was crushed using a beadbeater and subsequently incubated for a minimum of 3 h at 60 C in 500 pi CTAB buffer to which 6 pi proteinase K (10 mg/ml) was added. Next, 250 pi 7.5 M ammonium acetate was added and the mixture was centrifuged at 14,000 rpm for 10 min. The supernatant was saved and DNA was precipitated by adding anequal volume of isopropanol. The sediment was washed with 70% ethanol and redissolved in 25 pi water. Small aliquots (usually 1 pi) were used as templatefor PCR. The complete region separating the SSU and LSU genes and comprising the ribosomal spacers ITS1 and ITS2, and the conserved 5.8S gene, was amplified using the polymerase chain reaction. The total length of the fragment sequenced amounted to 712 bp. Finding primer pairs that yielded sequenceableamplicons was a tedious task. In all, we used the following primers: Vrain 2f; 5 -CTT TGT ACA CAC CGC CCG TCG CT- 3 Ferris 2f : 5 -RGY AAA AGT CGT AAC AAG GT- 3 Vrain 2r: 5 -TTT CAC TCG CCG TTA CTA AGG GAA TC- 3 28R1 : 5 - TGA TAT GCT TAA NTT CAG CGG GT fl : 5 - TCG AAT TGT GAA CTG CAG GAC ACA T r3 ; 5 - TCC GTG GGC TGC AAT GTGCGT TCG AA - 3 The PCR conditions were 30 s at 94 C, 30 s at 54 C and 2 min at 72 C for 40 cycles. After treatment with shrimp alkaline phosphatase and exonuclease I the PCR products could be used as template for cycle sequencing without further purification. PHYLOGENETIC ANALYSIS - DNA sequences were alignedwith MUSCLE (EDGAR, 2004) using default settings. Analyses were performed under unweightedparsimony,maximum likelihood and Bayesian inference. Trees were displayedwith TREEVIEW 1.6.6(PAGE, 1996). Parsimony analysis was performed using PAUP 4.0W0(SWOFFORD, 2003) with the followingheuristic search settings: 100 random taxon addition replicates, followed by tree-bisection-reconnection branch (TBR) swapping (10 7 rearrangements). Gaps were treated as missing data. Nodal support assessed was by calculating bootstrap values from 100 bootstrap replicates obtained by heuristic search with 10 ran-

4 368 H.J. Dumont, A. Vierstraete & J.R. Vanfleteren dom sequence additions each (FELSENSTEIN, 1985). Maximum likelihood analysis was performed using the generaltime-reversible substitution model with a gamma correction for among-site variation and corrected for invariable sites (GTR + I + G). This model was identified asthe best-fit model of DNA evolution formaximum likelihood analysis of the data-set by the likelihood-ratio test (LRT) and Akaike Information Criteria (AIC), implemented in ModelTest 3.7 (POSSADA & CRANDALL, 1998). Maximum likelihood was performed using the heuristic search option with stepwise taxon addition, TBR branch swapping, MulTrees option in effect, no steepest descent, and rearrangements limited to Bootstrapvalues were determined from 100 bootstrap replicates by heuristic search with 10 random sequence addition replicates each (FELSENSTEIN, 1985). Bayesian analysis was performedusing MrBayes, version (HUELSENBECK& RONQUIST, 2001). MrModeltest 2.2 (NYLANDER, 2004) also identified GTR + I + G as the best-fit model for Bayesian inference. The parameters for base frequencies, substitution rate matrix, gamma rate distribution and shape and proportion of invariant sites were allowed to vary throughout the analysis. Fig. 1. Bayesian probability estimate of the phylogeny of the Calopteryginaebased on internal Iran scribed spacer (ITS 1 and 2) data.

5 Revised molecular phytogeny of Calopteryginae 369 The Markov chain Monte Carlo was overfour process run parallelchains (one cold and three heated) for 1,000,000 generations with trees being sampled every 100 generations.the burn-in value was set to 1000 trees (i.e. 100,000 generations)equating the number of generations needed to reach a stable value of all variable parameters in a preliminaryrun. Majority rule consensustrees were reconstructed after discarding the burn-in. RESULTS We obtained the unambiguous sequences of the ITSes of all taxa listed in Table I, from which we computed four phylogenetic trees. The length of ITS varied from 163 to 203 bp, thatof ITS2 from 206 to 229 bp, and thatof 5.8 S from 163 to 166 bp. The Bayesian and maximum-likelihood-basedtrees (heuristic tree only) are shown in Figs 1 and 2. Four most parsimonious trees were found, differing only Fig. 2. Maximum likelihood estimate of the phylogeny of the Calopteryginae based on internal transcribed spacer (ITS1 and 2) data. Bootstrap support based on 100 replicates and expressed as a percentage.

6 - [The 370 H.J. Dumont, A. Vierstraete & J.R. Vanfieteren in minordetails (the branching order of species within the genus Calopteryx). Only the consensus tree is shown (Fig 3). We also derived a neighbor-joining tree, but since this fully confirmed the previous it ones, is not reproduced here. The Caloptery- into two subclades. The vast majority occur in East and South-East Asia; only cluster 1 ginae form three well-defined clusters, each of which can be subdivided {Calopteryx s. s.) extends west across the southern halfof Fig. 3. Heuristic Maximum Parsimony tree with branch lengths of the phylogeny of the Calopteryginae based on internal transcribed spacer (ITS 1 and 2) data. The MP analysis generated 4 most parsimonioustrees of 1118 steps each. scale bar represents 10 steps] Siberia and central Asia to reach Europe and the Mediterranean basin. Cluster 1 also has a subcluster that occurs in Canada and the USA. DISCUSSION Most branches of our trees are well supported, allowing some rather robust conclusions to be drawn. Weaker support is only found in the relationship between two Asian genera, Matronaand Atrocalopteryx, and some uncertainty also remains as to the descendenceorder within the genus Atrocalopteryx. All tree topologies reveal an undisputed monophyletic origin for the Calopteryginae (Bayesian probability 1.00; Maximum Parsimony, Maximum likelihood, and Neighbor joining all supported by 100% of the bootstraps executed), as established earlier by DUMONT et al. (2005), and the threemain clusters foundin

7 Revised molecular phylogeny of Calopteryginae 371 that paper are also confirmed (Figs 1-3). However, some important internal shifts fall to be recorded. Archineura incarnata is now closely clustering with Leucopteryx haeterinoides, forming a subclade that is well supported (Bayesian probability 0.99), as well as showing a close affinity between its two members (BP 1.00). The generic status of Archineura is thus accepted, and the needfor a separate genus ( Leucopteryx ) for taxon hetaerinoides lapses. New findings also include the fact that the position ofarchineurais now fully embeddedwithinincluster 3, and shows a sister-group relationship with the threeother genera that consitutethat cluster. Its relationship with such genera as Calopteryx, Matrona, and Atrocalopteryx is comparatively remote, not close as claimed in DUMONT et al. (2005). Cluster 2 is now betterstructured, with the clade Neurobasis-Matronoidesin sister position to a clade composed of Matrona and Atrocalopteryx. Matronacomes out of the analysis as a coherent, monophyletic genus, that is clearly descended from an Atrocalopteryx-like ancestor. Therelationships within Atrocalopteryx are less straightforward, although clearly, Calopteryx coomaniis seen to belong in Atrocalopteryx or a closely related taxon, and could well be the closest ancestor to Matrona. It certainly is not a true Calopteryx and this position is supported, by all three analyses independently. Cluster 2 is also characterized by only South-East Asiatic taxa, and suggests local evolution in the absence of major environmental disturbances such as glaciations. This involves quite a bit of cryptic speciation, well illustrated by Matrona, where the basilaris-group is clearly composed of several species that have so far not been well characterized morphologically (e.g. a taxon, provisionally called basilaris, on the island of Hainan). In cluster 2b, the endemic Matronoidesof Borneo is behaving as a genus distinct from Neurobasis, with the Indian specimen of Neurobasis chinensis suggesting at least evolution at the subspecies level withinthis widespread oriental taxon. It will be interesting to add to the analysis representatives of Neurobasis from Borneo, Sulawesi and The Phillipines, where this genus is rather speciose, to further refine the relationships within this cluster. Cluster 2 furthermore raises the problem whether true Calopteryx, a genus of temperate and continentaleurasia and North America, really occurs south of the latitude of Japan. The little-knowntaxa C. melli, C. oberthueriand C. laosica from South China, Laos and Vietnam may well, like coomani, turn out to belong to different, still undescribed, genera. Cluster 1 (Calopteryx) and its two subclusters (an Eurasian and a North American one) remain unaltered as compared to the analysis of DUMONT et al. (2005), as well as the genera composing cluster 3 for the addition of (except Archineura).

8 372 H.J. Dumont, A. Vierstraete & J.R. Vanfleteren CONCLUSION The subfamily Calopteryginae is composed of predominantly Asian representatives. A basal cluster of four genera ( Psolodesmus, Echo, Mnais, and Archineura) is currently restricted to subtropical-tropical East Asia. A second cluster (four genera, but probably one or two remaining to be defined) extends from the Asian tropics to temperateeast Asia. Calopteryx, finally, is a monogeneric cluster that is relatively species-poor and the only component of the Calopteryginae that extends through North-Central Asia to Europe, reaching North Africa in the west, invading North America in the East. ACKNOWLEDGEMENTS We thank all colleagues who provided the material necessary for this study. MATTI HAmAlAIN- EN provided material and gave invaluable advice about a number of taxa. PETER WEEKERS technical help with DNA extraction, amplification,and sequencing is appreciated. ROPING HAN made it possible for the senior author to visit Guangdong Province and Hainan Island (China), and collect a number of critical taxa there. REFERENCES DUMONT, H.J., J.R. VANFLETEREN, J.F. DE JONCKHEERE & P.H.H. WEEKERS, 2005, Phylogenetic relationships, divergence time estimation, and globalbiogeographic patterns of Calopterygoid damselflies (Odonata, Zygoptera) inferred from ribosomal DNA sequences. Sysl. Biol. 54: EDGAR, R.C., MUSCLE: multiple sequence alignmentwith high accuracy and high throughput. Nucleic Acids Res. 32: FELSENSTEIN, J., Confidence limits onphylogenies; anapproach using the bootstrap. Evolution 39: HUELSENBECK, J.P. & F.R. RONQUIST, MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics 17: KOCHER, T.D., W.K. THOMAS, A. MEYER, S.V. EDWARDS, S. PAABO, F.X. VILLABLANCA & A.C. WILSON, Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc. natn Acad. Sci. USA 86: NYLANDER, J.A.A., MrModeltest v2. Program distributed by the author. Evolutionary Biol Centre, Uppsala Univ. PAGE, R.D.M., TREEVIEW: An application to display phylogenetic trees on personal computers. Computer Applies Biosci. 12: POSADA, D. & K.A. CRANDALL, Modeltest: testingthe model of DNA substitution. Bioinformatics 14: SWOFFORD, D.L., PAUP RM : Phylogeneticanalysis usingparsimony ( RM and other methods) [Version 4], Sinauer Associates, Sunderland,Massachusetts. THOMPSON, J.D., D.G. HIGGINS & T.J. GIBSON, CLUSTAL W: Improving the sensitivity of progressive multiplesequence alignmentthrough sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: